The present invention relates to identifying novel processes involved in mediating arterial remodeling.
Arterial stenosis with reduction in blood flow is a common problem in many vascular diseases. Several growth factors have been implicated in the mechanisms leading to vascular stenosis. For instance, fibroblast growth factor 2 (FGF-2) has been identified as an important factor in mediating proliferation of smooth muscle cells leading to intimal lesion formation. Furthermore, it has been demonstrated that arterial stenosis in response to angioplasty is largely due to negative remodeling as a result of adventitial fibrosis. As more fully set forth below, transforming growth factor beta (TGF-xcex2) signaling has been demonstrated to play an important role in arterial stenosis in that, among other things, inhibition of TGF-xcex2 signaling using a soluble TGF-xcex2 receptor type II dramatically reduced lumen narrowing by decreasing negative remodeling and adventitial matrix deposition as well by decreasing neointima formation. These results indicate the crucial role of TGF-xcex2 signaling in arterial response to injury.
Vascular remodeling is a response of blood vessels to both physiological and pathological stimuli, leading to either vessel enlargement (positive remodeling) or shrinkage (negative remodeling). It has been demonstrated that neointimal proliferation or intimal mass following angioplasty shows little correlation with restenosis because of permanent changes in vascular geometry (Kakuta et al., 1994, Circulation 89:2809-2815; Nunes et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:156-165). Negative remodeling has been shown to account for most of the restenosis process (Mintz et al., 1993, Circulation 88:1-654), and is now generally considered the predominant cause of restenosis. A successful therapeutic approach to restenosis, therefore, would target negative vascular remodeling.
Several growth factors have been implicated in the mechanisms leading to vascular stenosis, such as fibroblast growth factor-2 (FGF-2) and transforming growth factor-xcex2 (TGF-xcex2). Specifically, cellular responses involving TGF-xcex2 in the adventitia have gained increased attention for their potential involvement in adventitial remodeling (Wilcox et al., 1996, Int. J. Radiat. Oncol. Biol. Phys. 36:789-796; Wilcox and Scott, 1996, Int. J. Cardiol. 54S:S21-35; Shi et al., 1996, Circulation 93:340-348). There is evidence that proliferative events occurring in the adventitia contribute to vascular remodeling and restenosis in response to vascular injury (Wilcox et al., 1996, Int. J. Radiat. Oncol. Biol. Phys. 36:789-796; Wilcox et al., 1997, Ann. N.Y. Acad. Sci. 811:437-447; Scott et al., 1996, Circulation 93:2178-2187). There is now general agreement that TGF-xcex2 is a potential factor in the adventitial remodeling process (Shi et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16:1298-1305).
Although it is known that the TGF-xcex2 family of cytokines can have a variety of effects on vascular cells, very little is known about the role of this family of cytokines in vascular remodeling. TGF-xcex2 affects many functions including proliferation of smooth muscle cells (SMC) (Halloran et al., 1995, Am. J. Surg. 170:193-197). It has been demonstrated that inhibition of SMC proliferation by TGF-xcex2 occurs via extension of the G2 phase of the cell cycle (Grainger et al., 1994, Biochem. J. 299:227-235). In contrast, it has also been shown that inhibition of SMC proliferation by TGF-xcex21 is due to arrest in the late G1 phase of the cell cycle (Reddy and Howe, 1993, J. Cell Physiol. 156:48-55). SMC derived from atherosclerotic lesions responded to TGF-xcex21 with an increase in proliferation, and lower levels of TGF-xcex2 receptor II (TGF-xcex2RII) have been implicated in the lack of inhibition by TGF-xcex2 in these cells (McCaffrey et al., 1995, J. Clin. Invest. 96:2667-2675).
Further studies have established that TGF-xcex21 stimulates SMC proliferation in vitro. Low doses of TGF-xcex21 stimulated SMC proliferation via platelet-derived growth factor (PDGF)-amino acid (AA)-dependent and PDGF-AA-independent mechanisms, while higher doses of TGF-xcex21 were inhibitory (Battegay et al., 1990, Cell 63:515-524; Stouffer and Owens, 1994, J. Clin. Invest. 93:2048-2055). Bifunctional effects of TGF-xcex21 in migration assays with SMC were also demonstrated (Koyama et al., 1990, Biochem. Biophys. Res. Commun. 169:725-729; Mii et al., 1993, Surgery 114:464-470).
TGF-xcex21 also plays a role in intimal lesion formation as indicated by a 5-7 fold induction of TGF-xcex21 mRNA in the balloon-injured rat carotid artery, with elevated levels of TGF-xcex21 mRNA persisting for 2 weeks (Majesky et al., 1991, J. Clin. Invest. 88:904-910). During the 2 week period, elevated TGF-xcex21 mRNA levels correlated with increases in mRNA expression of fibronectin and alpha-2 (I) and alpha-1 (III) collagens. These studies also demonstrated that infusion of recombinant TGF-xcex21 caused an increase in intimal SMC proliferation in vivo (id.).
Among clinically significant findings regarding the role of TGF-xcex2 signaling in arterial response to injury, it has been demonstrated that TGF-xcex21 mRNA expression in restenotic lesions compared to primary atherosclerotic lesions is increased (Nikol et al., 1992, J. Clin. Invest. 90:1582-1592). In the rat balloon injury model, treatment with TGF-xcex21 antibodies caused a small but significant reduction in neointima formation (Wolf et al., 1994, J. Clin. Invest. 93:1172-1178). Overexpression of TGF-xcex21 in the rat carotid artery by adenoviral gene transfer led to transient neointima formation with cartilaginous metaplasia that almost completely resolved within 8 weeks (Shulick et al., 1998, Proc. Natl. Acad. Sci. USA 95:6983-6988). Without wishing to be bound by any particular theory, TGF-xcex21 may also effect vascular tone since the factor was found to suppress nitric oxide synthase expression (Perella et al., 1996, J. Biol. Chem. 271:13776-13780) while at the same time inducing the vasoconstrictor endothelin in SMC in vitro (Kurihara et al., 1989, Biochem. Biophys. Res. Commun. 159:1435-1440). Further, TGF-xcex21 has been implicated in anti-apoptotic effects in SMC (Herbert and Carmeliet, 1997, FEBS Lett. 413:401-404).
Studies examining the expression of TGF-xcex2 ligand and TGF-xcex2 receptor (TGF-xcex2R) mRNAs using reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that TGF-xcex21, TGF-xcex23, and TGF-xcex2RII mRNA levels were increased in the media of the injured rat carotid artery (Ward et al., 1997, Arterioscler. Thromb. Vasc. Biol. 17:2461-2470) and expression of TGF-xcex22 and TGF-xcex23 were also reported in SMC of the lung vasculature (Khalil et al., 1996, Am. J. Respir. Cell Mol. Biol. 14:131-138; Pelton et al., 1991, Am. J. Respir. Cell Mol. Biol. 5:522-530). However, reduced levels of TGF-xcex2RII were demonstrated in human atherosclerotic lesions (McCaffrey et al., 1995, J. Clin. Invest. 96:2667-2675). The three TGF-xcex2 ligands have overlapping functions and all of them induce expression of the alpha-1 (I), alpha-2 (I) and alpha-1 (III) chains of collagen (Bray et al., 1998, Hypertension 31:986-994).
The role of TGF-xcex2 isoforms in vascular repair processes was examined using a rat balloon catheter denudation model (Smith et al., 1999, Circ. Res. 84:1212-1222). Proliferating and quiescent SMC in denuded vessels expressed high levels of mRNA for TGF-xcex21, TGF-xcex22, and TGF-xcex23, and lower levels of TGF-xcex2RII mRNA (Smith et al., 1999, Circ. Res. 84:1212-1222). The role of TGF-xcex2 signaling in the rat carotid artery balloon injury model was tested and it was shown that control vessels developed an extensive neointima and adventitial fibrosis with abundant collagen production. Vessels from animals injected with a recombinant soluble TGF-xcex2RII (designated as xe2x80x9cTGF-xcex2R:Fcxe2x80x9d) revealed only little neointima formation and much less collagen deposition in the adventitia. The adventitia also contained significantly fewer cells, indicating that the proliferation of adventitial fibroblasts is mediated by TGF-xcex2. Further, inhibition of TGF-xcex2 signaling with TGF-xcex2R:Fc dramatically reduced lumen narrowing by decreasing negative remodeling and adventitial matrix deposition, as well as neointima formation.
TGF-xcex2 has been implicated in myofibroblastic transdifferentiation (Orlandi et al., 1994, Exp. Cell Res. 214:528-536; Desmouliere et al., 1993, J. Cell Biol. 122:103-111; Verbeek et al., 1994, Am. J. Pathol. 144:372-382), causing fibroblasts to transiently express smooth muscle xcex1-actin (Darby et al., 1990, Lab. Invest. 63:21-29). The expression of smooth muscle xcex1-actin in the carotid artery was examined using immunostaining at 4 days after balloon denudation when proliferation of adventitial fibroblasts is rapid. Immunoreactive smooth muscle xcex1-actin was either completely absent or markedly reduced in the outer adventitia of vessels from rats treated with TGF-xcex2R:Fc compared to controls. This result demonstrated that the induction of smooth muscle xcex1-actin expression by adventitial fibroblasts is at least in part mediated by TGF-xcex2.
Morphometric analysis of the carotid arteries demonstrated significant increases in lumen area in all rats treated with TGF-xcex2R:Fc with an approximate 88% increase with a dose of 2 mg/kg given every other day for 2 weeks. Further, a dose of 0.5 mg/kg every other day for 2 weeks caused nearly a 60% increase in lumen area despite the fact that intimal lesion formation was not affected by this dose. These results indicate that loss of lumen area is in large part due to negative remodeling and measurements of the perimeter of the neointima (IEL) and media (EEL) demonstrated that all doses of TGF-xcex2R:Fc used in this study significantly inhibited the reduction in IEL and EEL.
The effect of TGF-xcex2R:Fc on remodeling is highly relevant to the clinical situation of restenosis after angioplasty (Mintz et al., 1993, Circulation 88:1-654; Mintz et al., 1994, Circulation 90:1-24). Immunostaining with anti-human IgG antibody demonstrated that the TGF-xcex2R:Fc primarily localized to the adventitia and neointima indicating that these are the predominant sites of TGF-xcex2 activity because TGF-xcex2R:Fc binds only active TGF-xcex2. One prominent effect of soluble TGF-xcex2RII was the effect on collagen synthesis, which was particularly striking in the adventitia of Masson""s trichrome stained sections. It was further found that the effects of TGF-xcex2R:Fc on collagen expression by Northern blot analysis of RNA isolated from carotid arteries 4 days after injury were markedly reduced for collagen Type I and Type III, but Type XV was unaffected. No differences in levels of osteopontin, tropoelastin, or fibronectin mRNA were detected.
Taken together, the aforementioned findings identify the TGF-xcex2 isoforms as major factors mediating adventitial fibrosis and negative remodeling following vascular injury. Thus, genes whose expression is affected by TGF-xcex2 are likely involved in such TGF-xcex2 associated processes, including arterial stenosis mediated by, inter alia, adventitial fibrosis and negative remodeling.
Arterial stenosis with reduction in blood flow is a common problem in many vascular diseases and it is an important causal factor in the morbidity and mortality associated with these diseases. Despite the fact that various growth factors, especially TGF-xcex2, have been implicated in arterial stenosis, very few factors involved in arterial stenosis have been identified and characterized. Nevertheless, the identification of such factors is crucial in the development of diagnostics and therapeutics for treatment of vascular diseases associated or mediated by arterial stenosis. Thus, there is long-felt need for the identification and characterization of factors associated with arterial stenosis. The present invention meets this need.
The invention includes an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof.
In one aspect, the nucleic acid shares at least about 33% sequence identity with a nucleic acid encoding at least one of rat REMODEL (SEQ ID NO:1), and a human REMODEL (SEQ ID NO:3).
The invention also includes an isolated nucleic acid encoding a mammalian REMODEL, wherein the amino acid sequence of the REMODEL shares at least about 6% sequence identity with an amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.
The invention includes an isolated polypeptide comprising a mammalian REMODEL. In one aspect, the mammalian REMODEL molecule shares at least about 6% sequence identity with an amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.
The invention includes an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof, wherein the nucleic acid further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto.
In one aspect, the tag polypeptide is selected from the group consisting of a green fluorescent protein tag polypeptide, an influenza virus hemagglutinin tag polypeptide, a myc tag polypeptide, a glutathione-S-transferase tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His6 tag polypeptide, a FLAG tag polypeptide, and a maltose binding protein tag polypeptide.
In another aspect, the nucleic acid further comprises a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
The invention includes a vector comprising an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof. In another aspect, the invention includes a recombinant cell comprising the vector. In a further aspect, the vector further comprises a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
The invention includes a recombinant cell comprising an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof.
The invention also includes an isolated nucleic acid complementary to an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof, the complementary nucleic acid being in an antisense orientation. In one aspect, the complementary nucleic acid shares at least about 33% identity with a nucleic acid complementary with a nucleic acid having the sequence of at least one of a rat REMODEL molecule (SEQ ID NO:1), and a human REMODEL molecule (SEQ ID NO:3).
The invention includes a recombinant cell comprising the isolated nucleic acid complementary to an isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof, the complementary nucleic acid being in an antisense orientation.
The invention includes an antibody that specifically binds with a mammalian REMODEL molecule polypeptide, or a fragment thereof.
In one aspect, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, and a synthetic antibody.
The invention includes a composition comprising the antibody that specifically binds with a mammalian REMODEL molecule polypeptide, or a fragment thereof, and a pharmaceutically-acceptable carrier.
The invention also includes a composition comprising the isolated nucleic acid complementary to the isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof, the complementary nucleic acid being in an antisense orientation, and a pharmaceutically-acceptable carrier.
The invention includes a composition comprising the isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof, and a pharmaceutically-acceptable carrier.
The invention includes a composition comprising the isolated polypeptide of comprising a mammalian REMODEL, and a pharmaceutically-acceptable carrier.
The invention includes a transgenic non-human mammal comprising the isolated nucleic acid encoding a mammalian REMODEL, or a fragment thereof.
The invention includes a method of treating a disease mediated by malexpression of a REMODEL molecule in a human. The method comprises administering to a human patient afflicted with a disease mediated by malexpression of a REMODEL molecule, a REMODEL molecule expression-inhibiting amount of the composition comprising the isolated nucleic acid complementary to the nucleic acid encoding a mammalian REMODEL, or a fragment thereof, and a pharmaceutically-acceptable carrier.
In one aspect, the disease is selected from the group consisting of impaired wound healing, fibrosis of an organ, ectopic ossification, and hypertrophic scar formation.
The invention includes a method of diagnosing arterial restenosis in a previously undiagnosed mammal. The method comprises obtaining a biological sample from the mammal, assessing the level of REMODEL in the biological sample, and comparing the level of REMODEL in the biological sample with the level of REMODEL in a biological sample obtained from a like mammal not afflicted with arterial restenosis, wherein a higher level of REMODEL in the biological sample from the mammal compared with the level of REMODEL in the biological sample from the like mammal is an indication that the mammal is afflicted with arterial restenosis, thereby diagnosing arterial restenosis in the previously undiagnosed mammal.
In one aspect, the biological sample is selected from the group consisting of a blood vessel sample, and a damaged tissue sample.
The invention includes a method of diagnosing negative remodeling in a previously undiagnosed mammal. The method comprises obtaining a biological sample from the mammal, assessing the level of REMODEL in the biological sample, and comparing the level of REMODEL in the biological sample with the level of REMODEL in a biological sample obtained from a like mammal not afflicted with negative remodeling, wherein a higher level of REMODEL in the biological sample from the mammal compared with the level of REMODEL in the biological sample from the like mammal is an indication that the mammal is afflicted with negative remodeling, thereby diagnosing negative remodeling in the previously undiagnosed mammal.
The invention includes a method of diagnosing fibrosis in a previously undiagnosed mammal. The method comprises obtaining a biological sample from the mammal, assessing the level of REMODEL in the biological sample, and comparing the level of REMODEL in the biological sample with the level of REMODEL in a biological sample obtained from a like mammal not afflicted with fibrosis, wherein a higher level of REMODEL in the biological sample from the mammal compared with the level of REMODEL in the biological sample from the like mammal is an indication that the mammal is afflicted with fibrosis, thereby diagnosing fibrosis in the previously undiagnosed mammal.
The invention includes a method of identifying a compound that affects expression of REMODEL in a cell. The method comprises contacting a cell with a test compound and comparing the level of REMODEL expression in the cell with the level of REMODEL expression in an otherwise identical cell not contacted with the test compound, wherein a higher or lower level of REMODEL expression in the cell contacted with the test compound compared with the level of REMODEL expression in the otherwise identical cell not contacted with the test compound is an indication that the test compound affects expression of REMODEL in a cell. In one aspect, the invention includes a compound identified by the method.
The invention includes a method of identifying a compound that reduces expression of REMODEL in a cell. The method comprises contacting a cell with a test compound and comparing the level of REMODEL expression in the cell with the level of REMODEL expression in an otherwise identical cell not contacted with the test compound, wherein a lower level of REMODEL expression in the cell contacted with the test compound compared with the level of REMODEL expression in the otherwise identical cell not contacted with the test compound is an indication that the test compound reduces expression of REMODEL in a cell. In one aspect, the invention includes a compound identified by the method.
The invention includes a method of identifying a compound that affects TGF-xcex2 signaling. The method comprises contacting a cell with a test compound and comparing the level of REMODEL expression in the cell with the level of REMODEL expression in an otherwise identical cell not contacted with the test compound, wherein a higher or lower level of REMODEL expression in the cell contacted with the test compound compared with the level of REMODEL expression in the otherwise identical cell not contacted with the test compound is an indication that the test compound affects TGF-xcex2 signaling in a cell.
The invention includes a kit for alleviating a disease mediated by malexpression of a REMODEL in a human. The kit comprises a REMODEL expression-inhibiting amount of a composition comprising the isolated nucleic acid complementary to the nucleic acid encoding a mammalian REMODEL, or a fragment thereof, and a pharmaceutically-acceptable carrier, the kit further comprising an applicator, and an instructional material for the use thereof. In one aspect, the disease is selected from the group consisting of negative remodeling, arterial restenosis, vessel injury, fibrosis.
The invention includes a kit for alleviating a disease mediated by malexpression of a REMODEL in a human. The kit comprises a REMODEL expression-inhibiting amount of the composition comprising encoding a mammalian REMODEL, or a fragment thereof, and a pharmaceutically-acceptable carrier, the kit further comprising an applicator, and an instructional material for the use thereof.